Krill protein hydrolysate reduces plasma triacylglycerol level with … · 2018-09-05 · The beneficial health effect of dietary krill intake has now been found in a limited number

Functional Foods in Health and Disease 2013; 3(11):428-440 Page 428 of 440

Research Article Open Access

Krill protein hydrolysate reduces plasma triacylglycerol level with

concurrent increase in plasma bile acid level and hepatic fatty acid

catabolism in high-fat fed mice

Marie S. Ramsvik1,2

, Bodil Bjørndal1, Rita Vik

1, Inge Bruheim

2, Jon Skorve

1, and Rolf

K. Berge1,3 *

1Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway;

2Olympic

Seafood AS, N-6080 Myrvaag, Norway; 3Department of Heart Disease, Haukeland

University Hospital, N-5021 Bergen, Norway

*Corresponding author: Rolf K. Berge, PhD, 1Department of Clinical Science, University

of Bergen, N-5020 Bergen, Norway

Submission date: September 10, 2013; Acceptance date: November 12, 2013; Publication

date: November 18, 2013

ABSTRACT

Background: Krill powder, consisting of both lipids and proteins, has been reported to

modulate hepatic lipid catabolism in animals. Fish protein hydrolysate diets have also been

reported to affect lipid metabolism and to elevate bile acid (BA) level in plasma. BA interacts

with a number of nuclear receptors and thus affects a variety of signaling pathways, including

very low density lipoprotein (VLDL) secretion. The aim of the present study was to

investigate whether a krill protein hydrolysate (KPH) could affect lipid and BA metabolism

in mice.

Method: C57BL/6 mice were fed a high-fat (21%, w/w) diet containing 20% crude protein

(w/w) as casein (control group) or KPH for 6 weeks. Lipids and fatty acid composition were

measured from plasma, enzyme activity and gene expression were analyzed from liver

samples, and BA was measured from plasma.

Results: The effect of dietary treatment with KPH resulted in reduced levels of plasma

triacylglycerols (TAG) and non-esterified fatty acids (NEFAs). The KPH treated mice had

also a marked increased plasma BA concentration. The increased plasma BA level was

associated with induction of genes related to membrane canalicular exporter proteins (Abcc2,

Abcb4) and to BA exporters to blood (Abcc3 and Abcc4). Of note, we observed a 2-fold

increased nuclear farnesoid X receptor (Fxr) mRNA levels in the liver of mice fed KPH. We

also observed increased activity of the nuclear peroxiosme proliferator-activated receptor

alpha (PPARα) target gene carnitine plamitoyltransferase 2 (CPT-2).

Conclusion: The KPH diet showed to influence lipid and BA metabolism in high-fat fed

mice. Moreover, increased mitochondrial fatty acid oxidation and elevation of BA

concentration may regulate the plasma level of TAGs and NEFAs.


Key words: Krill protein hydrolysate, triacylglycerol, fatty acids, TNFα

INTRODUCTION

Bile acids (BA), essential components for solubilization of dietary fats, are synthesized in the

liver from cholesterol. After synthesis, they are secreted across the canalicular membrane into

the biliary tree and finally end up in the gall bladder. From the gall bladder the BAs are

drained into the small intestine, reabsorbed by entrocytes and secreted into portal vein in

order to be returned to the liver to complete the enterohepatic cycle.

BAs interact with a number of nuclear receptors to stimulate a variety of signalling

pathways. One of these receptors is the nuclear receptor farnesoid X receptor (FXR). The

main physiological role of FXR is to maintain the BA homeostasis, and activation of FXR by

BAs will regulate genes encoding proteins involved in the enterohepatic cycle. As BAs are

reported to reduce plasma and liver triacylglycerol (TAG) level in animals [1, 2], this implies

that FXR also play a role in the control of lipoprotein metabolism and in the secretion of very

low density lipoprotein (VLDL) TAG lipoprotein fractions. Further, FXR also controls

plasma TAG levels by activating the nuclear peroxiosme proliferator-activated receptor alpha

(PPARα) gene [3]. PPARα is reported to induce genes encoding enzymes involved in

mitochondrial and peroxiosmal fatty acid oxidation (reviewed in [4]), and studies have shown

that modulation of the fatty acid oxidation capacity is important in regulation of the TAG

concentrations in serum and liver (reviewed in [5, 6]).

High plasma TAG concentration is a characteristic feature of the metabolic syndrome,

and our recent findings suggest that the hypotriacylglyceridemic property of PPARα

activation is primary due to effects on TAG synthesis and on mitochondrial fatty acid

oxidation. Abnormalities in insulin signaling have also been found associated with the

metabolic syndrome, and interestingly, glucose metabolism also seems to be regulated by

BAs and FXR activation (reviewed in [7, 8]). Furthermore, there is an increasing body of

evidence that PPARα does not only regulate lipid metabolism, but also the amino acid

metabolism [9, 10].

The beneficial health effect of dietary krill intake has now been found in a limited

number of studies. The hypotriacylglycerolemia has been attributed mostly to a greater

proportion of n-3 polyunsaturated fatty acids (PUFAs) in the phospholipid fraction of krill oil

[11, 12]. Recently, we reported that krill powder, which contains approximately 40% protein

(w/w) and 60% fat, increased liver lipid catabolism in tumor necrosis factor alpha (TNFα)

transgenic mice fed a high-fat diet [13]. Thus, the hypolipidemic effect of dietary krill extract

could be due to the oil and/or the protein content. It has been found that a krill peptide

showed blood-pressure lowering effects in animals [14], and a water-soluble extract of krill

prevented TAG accumulation in adipocytes in culture via suppression of PPARα activity

[15].

In the present study, we investigated whether the dietary water-soluble protein krill

protein hydrolysate (KPH) was able to affect hepatic lipid and BA metabolism, and to lower

plasma TAG levels in mice fed a high-fat diet for 6 weeks. We found that mice fed KPH

displayed increased mitochondrial fatty acid oxidation capacity and lower plasma TAG

concentration. Moreover, KPH-treated mice had a 16-fold higher fasting plasma BA levels

than mice fed casein.

MATERIALS AND METHODS


Animals and diets

Male C57BL/6 mice were used (Taconic, Germantown, USA). The experiments were

performed in accordance with, and under the approval of, the Norwegian State Board for

Biological Experiments, the Guide for the Care and Use of Laboratory Animals, and the

Guidelines of the Animal Welfare Act. Mice aged between 6 and 8 weeks were random

divided into a experimental (n = 6) and control (n = 9) group, and housed in pairs in open

cages with constant temperature (22 ± 2°C) and humidity (55 ± 5 %). They were exposed to a

12 h light-dark cycle and had unrestricted access to tap water and food. The mice were

acclimatized under these conditions with standard chow for one week prior the start of the

experiment. The animals weighed 27 ± 2 g at baseline, and there was no statistical difference

in the weight between the groups (data not shown).

Mice were fed ad libitum for 6 weeks on a high-fat control diet or a high-fat KPH

supplemented diet. The diets contained 20% (w/w) crude protein (N x 6,25) in the form of

bovine milk casein (Dyets Inc., Bethlehem, PA, USA) or as the water-soluble protein KPH

(Olympic Seafood AS, Myrvaag, Norway) (>70% with a molecular weight <1000 Da). All

diets contained 21% (w/w) fat consisting of lard (19%, a generous gift from Ten Kate Vetten

BV, Musselkanaal, Netherlands), and soy oil (2%, Dyets Inc.). The other constituents of the

diets were cornstarch, dyetrose, sucrose, fiber, AIN-93G mineral mix, AIN-93 vitamin mix,

L-Cysteine, Choline bitartrate (all from Dyets Inc.), and tert-Butyl-hydroquinone (Sigma-

Aldrich Norway AS, Oslo, Norway). The nutrition composition of the experimental diets is

described in Table 1, and the amino acid composition of casein and KPH is given in Table 2.

After 6 weeks of diet treatment, the mice were anaesthetized under non-fasting

conditions by inhalation of 2% isoflurane (Schering-Plough, Kent, UK). Blood was collected

by aortic puncture with 7.5% EDTA and immediately chilled on ice for a minimum of 15

minutes. The samples were centrifuged and plasma was stored at -80C prior to analysis.

Parts of the liver were chilled on ice and used for enzyme activities, and the rest were freeze-

clamped and stored at -80C until further analysis.

Plasma lipids and fatty acid composition

Plasma lipids were extracted according to Bligh and Dyer [16], evaporated under nitrogen,

and redissolved in isopropanol before analysis. Lipids from plasma were then measured

enzymatically on a Hitachi 917 system (Roche Diagnostics GmbH, Mannheim, Germany),

using the triacylglycerol (GPO-PAP) and cholesterol (CHOD-PAP) kit from Roche

Diagnostics, and the non-esterified fatty acid (NEFA) and phospholipid kit (phospholipids

FS) from DiaSys (Diagnostic Laboratories GmbH, Holzheim, Germany).

Table 1. Composition of the experimental diets*

Macronutrients Control (g/kg diet) KPH (g/kg diet)

Casein 224,7

KPH 266,7

Soybean oil 20 20

Lard 190 190

Cornstarch 90 90

Dyetrose 132 132

Sucrose 100 100


Fiber 50 50

Micronutrients

AIN-93G-MX mineral

mix

35 35

AIN-93VX vitamin

mix

10 10

L-cystine 3 3

Choline bitartrate 2,5 2,5

Tert-Butyl-

hydroquinone

0,014 0,014

KPH, krill protein hydrolysate.

*The diets were isonitrogenous and isoenergetic, and contained 20 g of crude protein/100 g

of diet. KPH consisted of 75% protein and 3% fat; casein consisted of 89% protein and 0,8%

fat.

Table 2. Amino acid composition in casein and krill protein hydrolysate

Amino acids AA Casein

(g/100 g protein)

KPH

(g/100 g protein)

Aspartic acid ASP 7,28 8,17

Glutamic acid GLU 22,00 12,02

Serine SER 5,44 3,45

Glycine GLY 1,80 5,67

Arginine ARG 3,25 8,29

Threonine THR 4,05 3,26

Alanine ALA 2,92 6,35

Proline PRO 10,48 8,03

Tyrosine TYR 4,22 5,36

Valine VAL 6,23 4,56

Methionine MET 2,58 2,88

Isoleucine ILE 4,85 4,84

Leucine LEU 9,08 8,20

Phenylalanine PHE 5,16 4,27

Lysine LYS 7,88 7,68

Histidine HIS 2,76 0,00

Taurine TAU 0,00 6,97

Methionine/Glycine MET/GLY 1,43 0,51

Lysine/Arginine LYS/ARG 2,43 0,93

KPH, krill protein hydrolysate.

Plasma bile acid measurement

BA from plasma were measured enzymatically on a Roche Modular P chemistry analyzer

(Roche Diagnostica), using the BA kit (Total Bile Acid Assy Kit, 05471605001) from

Diazyme (Diazyme Laboratories, Gregg, USA).


Hepatic enzyme activities

The livers were homogenized and a post-nuclear fraction was prepared as described earlier

[17]. The activities of carnitine palmitoyltransferase 1 (CPT-1a, EC number 2.3.1.21) and

carnitine plamitoyltransferase 2 (CPT-2, EC number 2.3.1.21), acyl-CoA oxidase 1,

palmitoyl (ACOX1, EC number 1.3.3.6), glycerol-3-phosphate acyltransferase (GPAT, EC

number 2.3.1.15) and fatty acid synthase (FASN, EC number 2.3.1.85) were measured in the

post-nuclear fraction as described by Skorve et al.[18].

Hepatic gene expression

Total cellular RNA was purified from frozen liver samples, and cDNA was produced as

previously described [19]. Real-time PCR was performed with Sarstedt 384 well multiply-

PCR Plates (Sarstedt Inc., Newton, NC, USA) on the following genes, using probes and

primers from Applied Biosystems: ATP-binding cassette, sub-family C, member 2 (Abcc2,

Mm00496899_m1), ATP-binding cassette, sub-family C, member 3 (Abcc3,

Mm00551550_m1), ATP-binding cassette, sub-family C, member 4 (Abcc4,

Mm01226381_m1), ATP-binding cassette, sub-family B, member 1b (Abcb1b,

Mm00440736), ATP-binding cassette, sub-family B, member 4 (Abcb4, Mm00435630_m1),

ATP-binding cassette, sub-family B, member 11 (Abcb11, Mm00445168), bile acid

Coenzyme A:amino acid N-acyl transferase (Baat, Mm00476075_m1), cytochrome P450,

family 7, sub-family A, polypeptide 1 (Cyp7a1, Mm00484152_m1), farnesyl X receptor

(Nr1H4, Mm00436419), solute carrier family 10, member 1 (Slc10a1, Mm00441421_m1)

and solute carrier organic anion transporter family, member 1b2 (Slco1b2,

Mm00451510_m1). Four different reference genes were included: 18S (Kit-FAM-TAMRA

(Reference RT-CKFT-18s)) from Eurogentec, Belgium, glyceraldehyde-3-phosphate

dehydrogenase (Gapdh, Mm99999915_g1) from Applied Biosystems, TATA box binding

protein (Tbp, AX-041188-00) and hypoxanthine guanine phosphoribosyl transferase (Hprt1,

AX-045271-00) from Thermo Fisher Scientific. Data normalized to 18S rRNA are presented.

Statistical analysis

Data sets were analyzed using Prism Software (Graph-Pad Software, version 6, San Diego,

CA) to determine statistical significance. The results are reported as means per group with

their standard deviations (SD). Due to the low number of observations in each group,

nonparametric statistical test were used. Mann-Whitney test was performed to evaluate

statistical differences between groups, and Wilcoxon signed-rank test was performed when

comparing matched pairs. P-values < 0.05 were considered significant.

RESULTS

Growth of mice fed krill protein hydrolysate

The average body weight in mice fed KPH decreased slightly over the test period (-2,3 ± 2,3

g, non significant.), whereas the mice fed casein gained weight (8,2 ± 4,0 g, p = 0,004),

resulting in a significant difference in change in weight between the groups over the period (p

= 0,0004) (Fig. 1a-c). The feed consumption was significantly higher in the KPH group

relative to the casein fed mice (p = 0,01) (Fig.1d). Further, the liver:body weight index was

significantly lower in the KPH mice than in control (4,1 ± 0,4 % in KPH vs. 4,7 ± 0,4 % in

control, p = 0,01) (Fig.1e).


C o n tro l K P H

-5

0

5

1 0

1 5

bo

dy

we

igh

t (

gra

ms

) ***

B a s e lin e E n d

1 5

2 0

2 5

3 0

3 5

4 0

4 5

K P H

bo

dy

we

igh

t (

g)

ns

B a s e lin e E n d

1 5

2 0

2 5

3 0

3 5

4 0

4 5

C o n tro l

bo

dy

we

igh

t (g

)

**

a b

cd

e

C o n tro l K P H

0

2

4

6

L iv e r in d e x

Liv

er:b

od

y w

eig

ht

(%) *

C o n tro l K P H

0

2

4

6

gra

ms

**

Figure 1. Body weight gain are reduced in mice fed a krill protein hydrolysate-based diet

Body weight gain in (a) the krill protein hydrolysate (KPH) group compared to (c) control (b) in male C57BL/6

mice. Food intake was measured as average intake between day 5-7, and day 17-19 (d). The liver:body weight

index (e) after six week of high-fat feeding containing casein or KPH. n = 6 for KPH groups and n = 9 for

control groups, except for measurements of food intake (d), in which n = 4 for KPH group and n = 6 for control.

Data in (c-e) are presented as mean ± SD. * Denote significant difference: Mann-Whitney test; Wilcoxon

signed-rank test (* = p < 0,05; ** = p < 0,01; *** = p < 0,001). KPH, krill protein hydrolysate; ns, non-

significant.

Reduced plasma lipids by feeding krill protein hydrolysate

The fasting plasma levels of TAGs, phospholipids and NEFAs were significantly reduced in

the KPH treated mice compared to control (p = 0,0007; p = 0,03; p = 0,03, respectively) (Fig.

2a-c). No difference was found in total plasma cholesterol (Fig. 2d).

Hepatic enzyme activities

Plasma TAG levels are determined by a balance between hepatic TAG synthesis and

secretion on one hand, and plasma TAG clearance on the other. Moreover, the hepatic fatty

acid oxidation capacity will regulate the VLDL synthesis and secretion. Interestingly, we

found that the activity of PPAR-α targeted gene CPT-2 was stimulated in KPH fed mice


compared to control (p = 0,015) (Fig. 3a). Higher activity of other PPAR-α targeted genes,

ACOX1 (p = 0,09) (Fig. 3b) and CPT-1 (in both absence and presence of malonyl-CoA), was

also found in KPH relative to casein fed mice, but the differences did not reach statistical

significance (CPT-1 with malonyl-CoA, p = 0,07; without malonyl-CoA, p = 0,15) (Fig. 3c).

Hepatic TAG biosynthesis and lipogenesis seemed to be unaffected by KPH treatment as the

activities of GPAT and FAS were similar in KPH treated mice compared to controls (Fig. 3d, e).

C o n tro l K P H

0

1

2

3

4

P la s m a C h o le s te r o l

mm

ol/

l

ns

C o n tro l K P H

0 .0

0 .5

1 .0

1 .5

P la s m a T A G

mm

ol/

l

***

C o n tro l K P H

0

1

2

3

4

P la s m a P h o s p h o lip id s

mm

ol/

l

*

C o n tro l K P H

-0 .1

0 .0

0 .1

0 .2

0 .3

P la s m a N E F A

mm

ol/

l

*

a b

c d

Figure 2. Krill protein hydrolysate reduces fasting plasma triacylglycerol

Concentrations of (a) triacylglycerol (TAG), (b) phospholipids, (c) non-esterified fatty acids (NEFA) and (d)

cholesterol in plasma of fasted male C57BL/6 mice fed high-fat diets containing casein or KPH. Data are

presented as mean ± SD, n = 6 for KPH groups and n = 8 for control groups. * Denote significant difference:

Mann-Whitney test (* = p < 0,05; *** = p < 0,001). KPH, krill protein hydrolysate; ns, non-significant; TAG,

triacylglycerol; NEFA, non-esterified fatty acid.

Plasma bile acid and hepatic genes of the enterohepatic cycle

It is reported that BAs have the potential to reduce plasma TAG levels [1, 2, 8, 20].

Therefore, it was of interest to note that KPH treatment increased the plasma BA level a 16-

fold compared to mice fed casein (Fig. 4).

BAs are synthesized in the liver from cholesterol. The expression of the rate-limiting

enzyme in this synthesis, Cyp7a1, was up-regulated in the liver of mice fed KPH. However, it

did not reach statistical significance (p = 0,06) (Fig. 5a). After BA is synthesized, it is

conjugated with glycine and taurine [8, 21]. Gene expression of Baat, responsible for this

conjugation, was not affected by KPH treatment compared to casein treated animals (Fig.

5b). The gene expression of Fxr however, important in maintaining the BA homeostasis, was

induced in KPH (p = 0,002) (Fig. 5c).


0

1

2

3

4

C P T -1 a c t iv ity

nm

ol/

mg

pro

tein

/min

ns

ns

C o n tro l K P H

w ith M a lo n y l C o A

w ith o u t M a lo n y l C o A

C o n tro l K P H

0

1

2

3

4

C P T -2 a c t iv ity

nm

ol/

mg

pro

tein

/min

*

C o n tro l K P H

0 .0

0 .2

0 .4

0 .6

0 .8

F A S a c tiv ity

mm

ol/

mg

pro

tein

/min ns

C o n tro l K P H

0

1

2

3

G P A T a c tiv ity

nm

ol/

mg

pro

tein

/min ns

a

c

d e

b

C o n tro l K P H

0

5

1 0

1 5

2 0

2 5

3 0

A C O X 1 a c tiv ity

mm

ol/

mg

pro

tein

/min

ns

Figure 3. The effect of krill protein hydrolysate on hepatic enzyme activity

Hepatic activities of (a) CPT-2, (b) ACOX1, (c) CPT-1, (d) GPAT, and (e) FAS in male C57BL/6 mice fed

high-fat diets containing casein or KPH. Data are presented as mean ± SD, n = 6. * Denote significant

difference: Mann-Whitney test (p < 0,05). ACOX1, acyl-CoA oxidase 1, palmitoyl; CPT-1, carnitine

plamitoyltransferase 1; CPT-2, carnitine plamitoyltransferase 2; FAS, fatty acid synthase; GPAT, glycerol-3-

phosphate acyltransferase; KPH, krill protein hydrolysate; ns, non-significant.

The enterohepatic cycle begins at the canalicular membrane of the hepatocyte, where

newly synthesized BAs are effluxed into the canaliculus. The gene expression of

Bsep/Abcb11, responsible for this transport, was down-regulated by KPH feeding (p = 0,002)

(Fig. 5d). Further, the expression of the cognate transporter Abcb4, important for bile flow,

was up-regulated by KPH feeding compared to the control (p = 0,004) (Fig. 5e). The mRNA

levels of Abcc2 was also increased after KPH treatment (p = 0,02) (Fig. 5f), whereas the gene

expression of Abcb1b was unaffected (Fig. 5g). No differences in expression of the BA

importer Slco1b2 were found (Fig. 5h), whereas the expression of Slc10a1/Ntcp was

significantly induced (p = 0,04) (Fig. 5i). Strikingly, the hepatic Abcc3 expression was

induced 2.4 fold after KPH treatment (p = 0,02) (Fig. 5j), and the expression of Abcc4 almost

11-fold (p = 0,002) (Fig. 5k).


C o n tro l K P H

-5

0

5

1 0

1 5

2 0

B ile a c id

µm

ol/

L

Figure 4. The effect of krill protein hydrolysate on plasma bile acid level

Concentrations of plasma bile acid (BA) in fasted male C57BL/6 mice fed high-fat diets containing casein or

KPH. Data are analyzed of pooled samples of four animals. BA, bile acid; KPH, krill protein hydrolysate.

C o n tro l K P H

0

5 0

1 0 0

1 5 0

C Y P 7 a 1

CY

P7

alf

a1

mR

NA

le

ve

l

ns

C o n tro l K P H

0

1

2

3

4

5

B A A T

BA

AT

mR

NA

le

ve

l

ns

C o n tro l K P H

0

1 0

2 0

3 0

4 0

F X R

FX

R m

RN

A l

ev

el **

C o n tro l K P H

0

1 0

2 0

3 0

4 0

5 0

A b c b 4

AB

CB

4 m

RN

A l

ev

el

**

C o n tro l K P H

0

1

2

3

B S E P /A b c b 1 1

AB

CB

11

mR

NA

le

ve

l **

C o n tro l K P H

0

2

4

6

A b c c 2

AB

CC

2 m

RN

A l

ev

el

*

C o n tro l K P H

0

1

2

3

A b c b 1 b

AB

CB

1B

mR

NA

le

ve

l

ns

C o n tro l K P H

0

1

2

3

4

S lc o 1 b 2

Slc

o1

b2

/OA

TP

4 m

RN

A l

ev

el

ns

C o n tro l K P H

0

2

4

6

8

S lc 1 0 a 1 /N T C P

Slc

10

a1

/NT

CP

mR

NA

le

ve

l

*

C o n tro l K P H

0

2 0

4 0

6 0

8 0

1 0 0

A b c c 3

AB

CC

3 m

RN

A l

ev

el *

C o n tro l K P H

-1 0

0

1 0

2 0

3 0

4 0

A b c c 4

AB

CC

4 m

RN

A l

ev

el

**

a b c

d e f

g h i

j k

Figure 5. The effect of krill protein hydrolysate on expression of hepatic genes in the

enterohepatic cycle. Hepatic gene expression of (a) Cyp7α1, the rate limiting enzyme in bile acid (BA)


synthesis, (b) Baat, responsible for conjugating BAs to taurine and glycine, and of (c) Fxr, important in

maintaining the BA homeostasis, in high-fat feeding containing casein or KPH. Gene expression of the

canalucular exporters (d) Bsep/Abcb11, (e) Abcb4, (f) Abcc2, (g) Abcb1b , the basolateral BA importers (h)

Slco1b2 , (i) Slc19a1/Ntcp, and the basolateral BA exporters (j) Abcc3 and (k) Abcc4. Values were related to the

reference gene 18S. Data are presented as mean ± SD, n = 6. * Denote significant difference: Mann-Whitney

test (* = p < 0,05; ** = p < 0,01). Abcb1b, ATP-binding cassette, sub-family B, member 1b; Abcb4, ATP-

binding cassette sub-family B, member 4; ABCC2, ATP-binding cassette sub-family C, member 2; Abcc3,

ATP-binding cassette, sub-family C, member 3; Abcc4, ATP-binding cassette, sub-family C, member 4; BA,

bile acid; Baat, bile acid coenzymeA:aminoacid N-acyltransferase; Bsep/Abcb11, bile salt export pump;

Cyp7a1, cytochrome P450, family 7, sub-family A, polypeptide 1; Fxr, farnesoid X receptor; Slco1b2, solute

carrier organic anion transporter family, member 1b2; Slc10a1/Ntcp , solute carrier family 10, member 1.

DISCUSSION

In the present study we have shown that a protein hydrolysate from krill elevated BA level,

with a concomitant reduction in plasma TAG concentration in high-fat fed C57BL/6 mice.

Moreover, induction of enzymes involved in mitochondrial fatty acid oxidation and genes

encoding proteins involved in BA synthesis, transport and secretion was found. Previously,

we reported that krill powder, consisting of both protein and fatty acids, increases liver lipid

catabolism in TNFa transgenic mice fed a high-fat diet [13]. These results are interesting, as

it appears that the bioactive component in these two dietary supplementations lie in the

protein fraction. The lipid lowering effect reported of KPH in the present study could be due

to its amino acid composition. Studies have suggested that dietary proteins with low ratios of

methionine/glycine and lysine/arginine have TAG-lowering effects [22]. As presented in

table 2, the amino acid composition in the two diets are different, and characterized by a

lower ratio of methionine/glycine and lysine/arginine in the KPH than in the casein control.

Further, the data suggest that BA metabolism cross-talks with lipid metabolism. Indeed,

BA administration has been shown to decrease plasma TAG concentration both in animals [1,

2] and humans with hypertriacylglycerolemia [23-25]. Further, increased concentration of

BAs most plausible induces transcription of Fxr target genes [26, 27]. FXR is not only an

important regulator for BA metabolism, but also for lipid metabolism [28]. FXR-deficient

mice have increased synthesis of Apolipoprotein B (ApoB), resulting in higher TAG

concentration in serum and liver [20]. It has also been reported that BA administration

induces VLDL receptor transcription levels via a FXR-dependent mechanism [29], and this

prevents elevated serum and liver TAG concentrations. Finally, BA treatment of mice

seemed to down-regulate lipogenesis via repression of SREBP-1C [2].

We found no significant differences in hepatic activities of GPAT and FAS in KPH-

treated mice compared to controls. Though, KPH treatment increased the hepatic fatty acid

catabolism as the activity of the PPARα target gene CPT-2 was increased. Further, the

hepatic gene expression of Fxr was increased a 2-fold in the KPH treated mice compared to

control. As FXR are reported to control plasma TAG levels by activating the PPARα gene

[3], and PPARα is reported to induce genes encoding enzymes involved in mitochondrial

fatty acid oxidation (reviewed in [4]), KPH regulation of BAs can possess the ability to

modulate hepatic fatty acid oxidation, leading to reduced plasma TAG concentration. These

results are in agreement with reported findings in rats by saithe Pollachius virens protein

hydrolysate [30] and salmon protein hydrolysate [31].

The increased BA concentration could be due to different factors, including BA

synthesis. Indeed, the gene expression of Cyp7a1 tended to be induced in KPH-treated mice.

The gene expression of the BA export pump Bsep/Abcb11 was down-regulated, and the


phosphatidylcholine floppase Aacb4, which efflux the main components of bile, was up-

regulated after KPH treatment. The expression of the canalicular exporter Abcb1b was

however unchanged, whereas the expression of the BA exporter to the blood Abcc4 was

induced by KPH feeding. Therefore, our data suggest that both higher BA synthesis and BA

secretion to blood are underlying factors for the elevated plasma BA concentration.

CONCLUSION

Dietary treatment with KPH resulted in lower plasma concentration of TAGs and NEFAs,

and elevated plasma level of BA compared to a casein control in high-fat fed mice. Further,

induction of enzymes involved in mitochondrial fatty acid oxidation, and genes encoding

proteins involved in BA synthesis, transport and secretion was found in the KPH-treated

group. Altogether, it is plausible to suggest that lipid and BA metabolism can be modulated

by protein hydrolysate derived from krill, and that increased mitochondrial fatty acid

oxidation and elevated BA concentration regulates plasma levels of TAGs and NEFAs. It is

unclear however, whether increased plasma BA levels are a cause or a response to

hypotriacylglycerolemia.

Abbreviations

ABC (ATP-binding cassette), BA (bile acid), ACOX (acyl-CoA oxidase), CPT (carnitine

palmitoyltransferase), CYP (cytochrome P450), FASN/FAS (fatty acid synthase), FXR

(farnesoid X receptor), GPAT (glycerol-3-phosphate acyltransferase), KPH (krill protein

hydrolysate), NEFA (non-esterified fatty acids), PPARα (peroxisome proliferator-activated

receptor alpha), SLC (solute carrier family), TAG (triacylglycerol), TNFα (tumor necrosis

factor alpha), VLDL (very low density lipoprotein.

Competing interests

The authors declare that they have no competing interests.

Author’s contributions

RKB and JS designed the research. BB, RKB and JS were involved in the acquisition of data.

MSR, RV, BB, RKB analyzed and interpreted the data. MSR, BB, IB and RKB wrote the

paper. All authors read and approved the final manuscript.

Acknowledgments and Funding

We thank to Kari Williams, Liv Kristine Øysæd, Randi Sandvik, Kari Helland Mortensen and

Svein Krüger for technical assistance, and Eline Milde and staff at the Laboratory Animal

Facility, University of Bergen, for care of the animals.

This project has been founded by NordForsk under the Nordic Centers of Excellence

programme in Food, Nutrition, and Health, Project (070010), MitoHealth and Olympic

Seafood AS. Olympic Seafood AS (Myrvaag, Norway) provided KPH, and Inge Bruheim and

Marie Sannes Ramsvik are employed at Olympic Seafood AS.

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Krill protein hydrolysate reduces plasma triacylglycerol level with … · 2018-09-05 · The beneficial health effect of dietary krill intake has now been found in a limited number